The specific hypothesis for this study is that there are fundamental differences in T effector and T regulatory cell responses in the lung to allergens in allergic asthma (AA) when compared to allergic nonasthmatics (ANA) that account for the difference in clinical responses. We will address this by comparing T cell responses in AA versus ANA subjects. These experiments will correlate T cell responses with measures of airway physiology using state-of-the art lung imaging and examine mechanisms controlling T cell activation in the airways of AA and the function of airway T regulatory cells during AA.

We will assess each BAL for inflammation and inflammatory mediator release. A pre-challenge BAL is obtained to insure the subject does not have baseline inflammation prior to the allergen challenge. The diluent challenged segment is used to control for effects of bronchoscopy and instillation of fluid in the lung. Analysis of the allergen challenged segment is done to confirm that allergen exposure led to airway inflammation, and allows us to correlate changes in airway physiology to inflammation in the allergen exposed segment.

A differential is calculated for each sample by counting at least 200 cells using morphology and staining characteristics to separate them into macrophages, lymphocytes, eosinophils, and neutrophils, allowing us to calculate the number and percentage of each cell type in the BAL. The cells are also stained for a panel of cell surface markers and intracytoplasmic proteins and analyzed by flow cytometry. This analysis will allow us to ensure that each subject responded to allergen challenge response with appropriate eosinophil and T cell recruitment into the BAL.

BAL fluid will be concentrated 10-fold using a Centricon filter (Millipore) with a 3,000 MW cutoff. We have found that assaying for cytokines is more reliable when the BAL is concentrated 10-fold since BAL is diluted ~100-fold by the procedure.Concentrations of a panel of 42 different cytokines, chemokines, and growth factors are measured using a LINCOplex human cytokine-chemokine kit as per manufacturer's guidelines (Millipore) and read on a Luminex 100 (Luminex Corporation). Results are analyzed using Beadview software (UpstateCell Signaling Solutions).

On the day of the first bronchoscopy,BAL will first be performed in the lingula without instillation of diluent or allergen.Then, a 2-ml aliquot of isotonic diluent is instilled into the right upper lobe. Then, the procedure will be repeated in the right middle lobe with instillation of 2-ml of standardized cat or mite allergen solution.A "test dose" concentration of allergen is administered first consisting of 2 ml of allergen at 1/10th(Cat,D.farinae) or 1/30th(D. pteronyssinus) the threshold concentration.If on visual inspection through the bronchoscope, there is no evidence of mucosal inflammation after two minutes, a second segmental allergen challenge will be done in the right middle lobe using 2-ml of full-dose allergen at the threshold concentration(Cat,D.farinae) or 1/3th the threshold concentration(D.pteronyssinus).This dose will be predetermined by quantitative skin prick testing.A second bronchoscopy is performed 24 hours after delivery of allergen extract and diluent.

Imaging is first performed the evening prior to the 1st bronchoscopy.An IV catheter is placed.An attenuation correction is performed to remove image distortion using a chest CT volumetric scan.Subjects are instructed to exhale to the same mean lung volume of the CT scan and hold their breath for 20sec.Simultaneous with apnea,13NNsaline is injected IV and a series of PET scans is acquired.Then subjects resume breathing, matching their previous respiratory rate and tidal volume.After 3 min,within an interval of 1 min,spirometry and 2 deep inhalations are performed,followed by 1 min of washout. For the second imaging visit, which will occur 24 hour later, the imaging sequence will be repeated as described above, but will also include 18FDG infusion.At least 30 minutes after the 13NN injection,10mCi of 18FDG is infused. Then, images are collected over a 75 minutes.Venous blood is sampled at 5 different time points over a 40-45 minute time window to determine plasma radioactive levels.

Other Names:

13N-N2 saline

18F-FDG, 0.019 mSv/MBq

Detailed Description:

Despite advances in medications, allergic diseases, including allergic asthma, continue to rise in prevalence. For this reason, there is a need for a better understanding of the mechanisms of allergic diseases and novel insights into modulating allergic inflammation. CD4+ Th2-type lymphocytes seems to be central to the pathogenesis of allergic disease, as the levels of these cells and Th2 cytokines (IL-4, IL-5 and IL-13) are elevated in the airways of allergic asthma patients. The unifying hypothesis of this project is that understanding the mechanisms that determine the critical balance of effector and regulatory allergen-specific T cell activity in asthma will lead to new approaches for inducing allergen-specific tolerance and new therapeutic strategies for asthma.

Eligibility

Ages Eligible for Study:

18 Years to 50 Years

Genders Eligible for Study:

Both

Accepts Healthy Volunteers:

No

Criteria

Inclusion Criteria:

Subjects with Allergic Asthma (AA subjects):

All subjects will have a baseline FEV1 no less than 75 % of the predicted value after bronchodilator administration.

All subjects will have both a clinical history of allergic symptoms to cat or dust mite allergen and a positive allergen prick test (3 mm diameter greater than diluent control)

Life-long absence of cigarette smoking (lifetime total of < 5 pack-years and none in 5 years).

Willing and able to give informed consent.

Expressed the desire to participate in an interview with the principal investigator.

Age between 18 and 50 years.

A methacholine PC20 < 16 mg/ml.

Asthma of severity defined as: requiring no more than step 3 therapy (NHLBI Guidelines, 2007 EPR-3, http://www.nhlbi.nih.gov/guidelines/asthma/asthgdln.pdf), well-controlled and having a validated asthma control test (ACT) score of > 19 for one month prior to the screening visit, and able to tolerate a 2 week stoppage of inhaled corticosteroids prior to Visit 2.

Allergic Nonasthmatic Subjects (ANA subjects):

ANA subjects will have a history of at least one of the following: (a) allergic rhinitis (with one or more of the following symptoms: nasal congestion, sneezing, runny nose, postnasal drainage), (b) allergic conjunctivitis (ocular itching, tearing and/or swelling) or (c) contact allergy associated with cat dander or dust mite and a positive allergy test to the same allergen.

All subjects will have a baseline FEV1 and FVC determined at the characterization visit that is no less than 90 % of the predicted value before bronchodilator administration.

All subjects will have a positive allergy skin prick test to cat dander or dust mite allergen.

All subjects will be in good general health.

Life-long absence of cigarette smoking (lifetime total of < 5 pack-years and none in 5 years).

Willing and able to give informed consent.

Expressed the desire to participate in an interview with the principal investigator.

Age between 18 and 50 years.

Exclusion Criteria:

Subjects with Allergic Asthma (AA subjects):

Women of childbearing potential who are pregnant (based on urine beta-HCG or STAT quantitative serum hCG testing), are sexually active and not using contraception, are seeking to become pregnant, or who are nursing.

The presence of spontaneous asthmatic episode or clinical evidence of upper respiratory tract infection within the previous 6 weeks.

Participation in a research study involving a drug or biologic during the 30 days prior to the study.

We will exclude severely and morbidly obese subjects (BMI> 32) because of the poor quality of images that can be obtained and weight restrictions on the scanner.

Those with a diffusing capacity < 80% predicted (if known),

Subjects with known exposure to agents that are associated with pulmonary disease (i.e. asbestos, silica)

Subjects who have had any research related radiation exposure greater than 15 mSv within the past year will be excluded.

Individuals with known allergy or hypersensitivity to FDG will be excluded.

Contacts and Locations

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Please refer to this study by its ClinicalTrials.gov identifier: NCT01612936